GSM6934214: CD45+Ter119- cells from bone marrow of Naïve BALB/c mice; Mus musculus; RNA-Seq
PB was collected by retro-orbital sampling. To isolate the BM cells, the muscle tissue on the tibia and femur were completely removed, BM cells were flushed out with PBS(2%FBS) from long bone (tibia and femur) and filtered through a 70 µm strainer. red blood cells are depleted by resuspending BM cells in RBC lysis buffer (Tonbo). For each group, cells were sampled from five mice and pooled into one, then stained with DAPI, CD45-APC and TER-119-VF450. To get rid of erythroid cells, DAPI-CD45+TER119- singlet cells were purified by fluorescence-activated cell sorting (FACS)orted into single-cell suspension and immediately submitted to the Single Cell Genomics Core at Baylor College of Medicine. The single-cell gene expression Library was prepared according to the Chromium Single Cell Gene Expression 3v3.1 kit (10x Genomics). In brief, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on the Chromium controller (10x Genomics) to generate single-cell GEMS (Gel Beads-In-Emulsions) where full-length cDNA was synthesized and barcoded for each single cell. Subsequently, the GEMS are broken and cDNA from each single cell is pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR. The amplified product is fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. The final library was generated by amplification. The libraries that passed the quality control, were sequenced by the NovaSeq 6000 (Illumina).